Erythrocytes containing arginine deiminase

ABSTRACT

Use of erythrocytes containing arginine deirainase for the preparation of a medicinal product for lowering the plasma concentration of arginine in vivo. The use relates in particular to the treatment of arginine-dependent tumors, such as hepatocarcinoma and malignant melanoma, or inhibition of the synthesis of nitric oxide, and the prevention and/or treatment of septic shock.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/912,236, filed Oct. 22, 2007, which was a 371 application ofInternational Application No. PCT/IB2006/001004, filed Apr. 25, 2006,all of said applications incorporated herein by reference.

The present invention relates to the depletion of plasma arginine, tocompositions for providing said depletion and to the treatment ofpathologies that may benefit from said depletion and its effects, forexample on the synthesis of nitric oxide. Thus, the invention relates tothe treatment of certain tumors, such as malignant melanoma andhepatocarcinoma, and to the prevention and treatment of septic shock.

All documents, articles and patents or patent applications cited hereinare hereby incorporated herein by reference.

Arginine is a nonessential amino acid. It is synthesized in the courseof the urea cycle, from citrulline in two stages, owing to the action ofargininosuccinate synthetase and argininosuccinate lyase. The firstenzyme catalyzes the conversion of citrulline to argininosuccinate andthe second performs the conversion to arginine. Arginine is metabolizedto ornithine under the action of arginase, and ornithine can in its turnbe transformed to citrulline by a reaction catalyzed by ornithinetranscarbamoylase.

It has been shown, however, that certain types of tumor cells requirearginine to be supplied, and this led to consideration of argininedepression as a possible treatment for these forms of cancers, calledarginine-auxotrophic. The antitumor activity of arginine deiminase hasbeen the subject of numerous publications. Thus, in vivo activity hasbeen demonstrated with respect to malignant melanoma andhepatocarcinoma. However, this enzyme has some major drawbacks.

Arginine deiminase is not produced in mammals but is obtained frommicroorganisms, making it a highly antigenic compound for a mammal.

Moreover, this enzyme has a very short half-life in mammals, of theorder of about 5 hours, and must be administered daily at a high dose tobecome effective.

To overcome these drawbacks, the authors proposed pegylated forms ofthis enzyme, i.e. arginine deiminase conjugated with polyethylene glycol(PEG), which led to less antigenic formulations with a longer half-life(from 7 to 9 days). Among works dealing with this subject, we maymention the following concerning the treatment of melanoma and ofhepatocarcinoma: F. Izzo et al., J. Clin. Oncol. 2004, 22: 1815-1822; C.M. Ensor et al., Cancer Research 2002, 62: 5443-5450; F. W. Holtsberg etal. J. Control. Release 2002, 80: 259-271; J. S. Bomalaski et al.,Preclinica, Research Article November/December 2003, 1, 5: 284-293;Curley S. A. et al., Hepatogastroenterology, 50, 1214-6, 2003.

U.S. Pat. No. 4,965,857 proposes an entirely different method, involvingextracorporeal treatment of the blood using a reactor in which argininedeiminase acts upon the extracorporeal circulation.

The current treatments based on pegylated arginine deiminase areinteresting, but have certain limitations connected with the need toadminister relatively large doses repeated at short intervals, as thehalf-life is still short, and with toxicity connected with repeatedadministration at high dose, in the long term with a risk of inducinglevels of antibodies to the active principle which may lead to allergiceffects and inhibition of the active principle.

It would therefore be of considerable benefit to have at our disposal anactive product having better bioavailability (better biologicalactivity, prolonged half-life), making it possible to optimize theamount of enzyme administered and lower the toxicity, even in the caseof repeated treatments, and limit the risks of immune reaction andclinical allergy.

The advantage of using red blood cells as vectors of medicinal productswas suggested long ago. They have natural biocompatibility and, aftertransfusion, they are completely biodegradable by a known process andhave a relatively long half-life in vivo (half-life of the order ofabout 30 days in man).

Encapsulation of arginase, a natural enzyme of the urea cycle, wasproposed and tested within the scope of treatment of hyperargininemicpatients exhibiting deficiency of this enzyme in their erythrocytes (C.G. Millan, J. Controlled Release 2004, 95: 27-49; K. Adriaenssens etal., Int. J. Biochem. 1984, 16, 7: 779-786). The aim was to compensatethe metabolic and enzymatic deficiencies connected with endogenousarginase deficiency.

Arginase has an alkaline optimum pH at about 9.5, and its activity islow at physiological pH. In contrast, arginine deiminase has an optimumpH of about 6.5, retains more than 70% of its activity at physiologicalpH, and its affinity for arginine is 1000 times greater than that ofarginase: B. J. Dillon et al., Med. Sci. Monit. 2002, 8, 7: 248-253. Inthat study, concerning inhibition of the synthesis of nitric oxide NO,the authors report strong activity of arginine deiminase onextracellular arginine, but absence of activity on intracellulararginine (macrophages).

NO is a biomediator and is thought to be synthesized essentially fromextracellular arginine. Septic shock is mediated by NO and by tumornecrosis factor TNFα. Inhibition of the synthesis of NO has beenenvisaged as a treatment against septic shock, hence the works of Dillonet al. supra and of J. B. Thomas, Biochem. J. 2002, 363, 581-587. NOalso seems to be involved in the cancerization process, as reported inLind D S. Arginine and cancer. J. Nutr. 2004: 2837S-41.

It appears that arginine deiminase possesses a potential for degradationof arginine and inhibition of NO synthesis far greater than arginase.However, this enzyme would not have intracellular enzymatic activity,the physiological pH is not its optimum pH, it has a short half-life andit is likely to induce an immune reaction.

Starting from this complex situation, the inventors set themselves theaim of proposing a technical solution that provides effectivedegradation of plasma arginine and/or inhibition of the synthesis ofnitric oxide NO.

Thus, the present invention relates to the use of erythrocytescontaining arginine deiminase for the preparation of a medicinal productfor lowering the concentration of plasma arginine in vivo.

Arginine deiminase is identified under reference EC 3.5.3.6 in IUBMBEnzyme Nomenclature. The enzyme employed can be of natural, synthetic orartificial origin, or obtained by genetic engineering (for exampleproduction of the enzyme in a host cell, for example E. coli, afterintegration of a vector expressing the gene coding for the enzyme).Arginine deiminases that can be used are described for example in EP-A-1011 717, EP-A-0 414 007, U.S. Pat. No. 5,372,942, JP-A-6062867,JP-A-2053490, JP-A-2035081. In an equivalent manner, the inventionincludes the use of analogues of this enzyme which can notably beenzymes that have been modified in order to increase their enzymaticactivity (EP-A-0 981 607).

The objective of the present invention is plasma arginine depletion,which means lowering the concentration of arginine in the plasma.Without wishing to be bound to a theory, it is thought that the plasmaarginine enters the treated erythrocytes by passive diffusion. Theerythrocytes of the invention behave as bioreactors, in which thearginine that enters is degraded by the arginine deiminase. Theinvention offers other advantages. At the end of their life, theerythrocytes are destroyed by macrophages, essentially in the liver, thespleen and the bone marrow, as well as in the lungs. This leads totargeted release of arginine deiminase, causing local depletion ofplasma arginine. This effect is utilized in the treatment ofpathologies, in particular of tumors, that affect these organs, such ashepatocarcinoma.

The solution adopted by the invention makes it possible to combineseveral decisive advantages in a remarkable way, namely the lifetime ofthe erythrocytes permitting a long time of action, storage of the enzymein an environment that is, on the one hand, favorable, with a pHgenerally below 7.4 at which the enzyme displays an enzymatic activitygreater than 80% and, on the other hand, preserved, since the enzyme isisolated from the immune system, thereby reducing the risk of developingan immune reaction to the enzyme, which is a great advantage in the caseof repeated treatments. The enzyme is protected from any anti-argininedeiminase antibodies by the membrane of the erythrocytes, and itsenzymatic activity is therefore preserved even when antibodies arepresent in the blood of the patient being treated. Furthermore, healthycells are preserved, as the enzyme does not act upon intracellulararginine.

The invention therefore finds particularly interesting application inarginine-dependent tumors, for which the favorable effect of plasmaarginine depletion has been demonstrated (see for example F. Izzo etal., 2004, C. M. Ensor et al., 2002, F. W. Holtsberg et al., 2002, J. S.Bomalaski et al., 2003, and Curley S. A. et al., 2003, previouslycited).

According to a first embodiment of the invention, the medicinal productis intended for the treatment of arginine-dependent tumors. Byarginine-dependent we mean tumors involving tumor cells that requirearginine for replication, are unable to synthesize some or all of thearginine that they need, and therefore require a supply of arginine.Plasma arginine depletion will deprive these cells of the arginine thatis essential for their development, leading to targeted death of thesecells, inhibition of tumor growth or regression of the tumor mass.

According to one characteristic, the invention relates to the use ofthese erythrocytes for the preparation of a medicinal product fortreating hepatocarcinoma or primary liver cancer.

According to a second characteristic, the invention relates to the useof these erythrocytes for the preparation of a medicinal product fortreating malignant melanoma, in its various forms, such as superficialspreading melanoma and nodular melanoma.

According to a second embodiment of the invention, the invention relatesto the use of these erythrocytes for the preparation of a medicinalproduct for inhibiting the synthesis of nitric oxide. It should bepointed out that the medicinal product acts at least partly via thedegradation of plasma arginine, as described in Dillon et al., 2002,cited previously.

According to one characteristic of this embodiment, the inventionrelates to the use of these erythrocytes for the preparation of amedicinal product for the prevention and/or treatment of septic shock.

The invention further relates to the use of these erythrocytes for thepreparation of a medicinal product for the treatment of one of thefollowing forms of cancer:

-   -   breast cancer (Shen Wei-Chiang et al., California Breast Cancer        Research Program);    -   neuroblastoma (Gong H. et al., Int. J. Cancer 2003, 106: 723-8);        and    -   leukemia (Gong H. et al. Leukemia 2000, Vol. 14, 826-9;        Noh E. J. et al., Int. J. Cancer 2004, 112: 502-8);        or for inhibition of angiogenesis and the treatment of        associated diseases such as: angioma, angiofibroma, arthritis,        diabetic retinopathy, retinopathy of the premature, neovascular        glaucoma, disease of the cornea, involutional and other forms of        macular degeneration, pterygium, retinal degeneration,        retrolental fibroplasia, psoriasis, telangiectasis, granuloma        pyogenicum, seborrheic dermatitis, acne, cancer and metastases        connected with angiogenesis (WO0209741; Park I. S. et al.,        Br. J. Cancer 2003, 89: 907-14).

The invention also relates to erythrocytes containing argininedeiminase.

The invention further relates to a suspension of these erythrocytes in apharmaceutically acceptable saline solution (generally, standard mediumfor erythrocytes, solution containing NaCl and one or more ingredientsselected from glucose, dextrose, adenine and mannitol; e.g. SAG-mannitolor ADsol). Said solution can provide preservation of the erythrocytes,and it can include a preservative such as L-carnitine. Said suspensioncan be packaged ready for use or for dilution before use. The finalhematocrit value of the ready-to-use product (after dilution before use,if necessary) is preferably between 40 and 70%. It can be administeredintravenously, preferably by perfusion.

Such a suspension or any administrable formulation containingerythrocytes according to the invention constitutes in itself amedicinal product or a pharmaceutical composition covered by theinvention. Said medicinal product or composition can notably be intendedfor the various applications mentioned above. It can be packaged forexample as a flexible bag for perfusion, or in some other form foradministration by injection.

According to one characteristic of the invention, the medicinal productcomprises a suspension of erythrocytes with a hematocrit value between40 and 70%, preferably between 45 and 55%, and more preferably of 50%.It is preferably packaged in a volume of 10 to 250 ml. The quantity ofencapsulated enzyme corresponding to the medical prescription ispreferably contained wholly in the bag of blood. A medical prescriptioncan vary from 1 to 200 IU per kg of body weight.

The invention also relates to a method of treatment ofarginine-dependent forms of cancer, such as hepatocarcinoma andmalignant melanoma, or one of the other cancerous or non-cancerouspathologies mentioned above, comprising the administration of aneffective amount of said medicinal product to a patient who needs it,notably by the intravenous route, by injection or perfusion, andpreferably by perfusion.

According to an interesting modality, the patient is treated aftersurgical excision of the tumor.

The invention also relates to a method of treatment intended to inhibitthe synthesis of nitric oxide and/or prevent and/or treat septic shock,comprising the administration of an effective amount of said medicinalproduct to a patient, notably by the intravenous route, by injection orperfusion, and preferably by perfusion.

According to a particularly advantageous modality for these variousmethods of treatment, the patient is treated with his own erythrocytes,after they have been treated for encapsulation of the enzyme. As avariant, the erythrocytes are obtained from one or more donors.

The method can thus comprise collecting one or more blood samples, forexample bag(s) of blood, from a patient or from one or more donors, thepreparation of a deposit or concentrate of erythrocytes, enzymeincorporation according to the invention and the production of a batchof erythrocytes incorporating the enzyme, then the administration of thesuspension (medicinal product) to the patient, by the intravenous route.

Typically, a volume of suspension of treated erythrocytes correspondingto from 1 to 200 IU of enzyme per kg of body weight is administered.According to one characteristic of the invention, from 10 to 250 ml of asuspension of erythrocytes at a hematocrit value between 40 and 70%,preferably between 45 and 55%, and more preferably of 50%, isadministered.

According to a particular modality, such a suspension is administered ata frequency between 15 days and three months, preferably monthly, for asufficient period of time.

The techniques for encapsulating active principles in erythrocytes areknown and the basic technique by lysis-resealing, which is preferredhere, is described in patents EP-A-101 341 and EP-A-679 101, to which aperson skilled in the art can refer. According to this technique, theprimary compartment of a dialyzer is supplied continuously with asuspension of erythrocytes, whereas the secondary compartment containsan aqueous solution that is hypotonic relative to the suspension oferythrocytes in order to lyse the erythrocytes; next, in a resealingunit, resealing of the erythrocytes is induced in the presence of theenzyme by increasing the osmotic and/or oncotic pressure, then asuspension of erythrocytes containing the enzyme is collected.

Among the variants described to date, preference will be given to themethod described in French patent application No. 0408667, whichprovides efficient, reproducible, reliable and stable encapsulation ofthe enzyme. This method comprises the following stages:

-   1—suspending packed red blood cells (PRBCs) (or globular    concentrate) in an isotonic solution at a hematocrit value greater    than or equal to 65%, refrigeration between +1 and +8° C.,-   2—measurement of osmotic fragility on a sample of erythrocytes from    the same PRBCs,-   where stages 1 and 2 can be performed in any order (including in    parallel),-   3—procedure for lysis and internalisation of the enzyme (in    particular within one and the same chamber), at a temperature    maintained constant between +1 and +8° C., comprising passing the    suspension of erythrocytes at a hematocrit value greater than or    equal to 65% and a hypotonic lysis solution refrigerated between +1    and 8° C. through a dialysis cartridge; the lysis parameters being    adjusted as a function of the osmotic fragility measured previously;    and-   4—resealing procedure (carried out in particular in a second    chamber) in which the temperature is between +30 and +40° C., in the    presence of a hypertonic solution.

“Internalisation” means penetration of the enzyme inside theerythrocytes.

According to a first characteristic of the invention, the PRBCs aresuspended in an isotonic solution at a high hematocrit value, greaterthan or equal to 65%, and preferably greater than or equal to 70%, andthis suspension is refrigerated between +1 and +8° C., preferablybetween +2 and +6° C., and typically at about +4° C. According to aparticular modality, the hematocrit value is between 65 and 80%, andpreferably between 70 and 80%.

According to an important characteristic of the invention, the osmoticfragility of the erythrocytes is measured just before the lysis stage.The erythrocytes or the suspension containing them are advantageously ata temperature close to or equal to the temperature chosen for lysis.According to another advantageous characteristic of the invention, oncethe measurement of osmotic fragility has been obtained it is quicklyutilized, i.e. the lysis procedure is carried out very soon after takingthe sample. This time delay between taking the sample and commencementof lysis is preferably less than or equal to 30 minutes, and even morepreferably less than or equal to 25 or even 20 minutes.

The two parameters permitting dialysis to be controlled are the dwelltime of the cells in the dialyzer (as a function of the characteristicsof the latter) and the osmolarity of the dialyzate. These two parametersmust be adjusted in relation to the characteristics of osmoticresistance, or conversely fragility, of the red blood cells which areprocessed for undergoing the stages of lysis/resealing. This osmoticresistance can be characterized by at least one of the followingparameters:

-   -   a. The osmolarity of the medium at which hemolysis appears, i.e.        the start of pore formation.    -   b. The rate V of hemolysis, determined from the slope of the        linear portion of the curve % hemolysis=f(osmolarity of the        medium).    -   c. The percentage hemolysis for a given osmolarity.    -   d. The osmolarity at which 50% hemolysis (H₅₀) is obtained.    -   e. The time taken to obtain a certain percentage of hemolysis        (for example 50%).

According to preferred embodiments, the osmotic resistance ischaracterized by means of the parameters b, d or b and d.

The osmotic fragility must therefore be measured in a short time,compatible with the short time delay between taking the sample andcommencement of lysis. According to one characteristic of the invention,one or more of these hemolysis parameters are measured against ahypotonic solution of known isotonicity, e.g. water (distilled wateretc.), through a semipermeable membrane. A manual method can beenvisaged. However, according to a preferred embodiment of theinvention, the osmotic fragility is measured using an automaticmeasuring instrument that is designed for measuring the osmoticfragility of a sample of erythrocytes in less than 15 minutes, moreparticularly in less than 12 minutes and preferably in less thanminutes, and the result obtained is utilized with a short time delay toadjust the lysis parameters, and begin lysis.

The osmotic fragility can be measured using an instrument thatautomates, at least in part, the manual technique described by J. V.Dacie in Practical Haematology, 2nd edition, Churchill, London 1956. Anexample of such an instrument is described in the article by J. Didelonet al., Clinical Hemorheology and Microcirculation 23 (2000) 31-42. Theprinciple is based on the use of a device in which the sample of thesuspension of erythrocytes to be evaluated, and a hypotonic solution ofknown isotonicity, e.g. distilled water, of suitable volumes, are placedon either side of a semipermeable membrane, so as to generate slowhemolysis of the erythrocytes as the NaCl ions diffuse towards thesolution, e.g. distilled water. The progress of hemolysis over time ismonitored by measuring the transmittance (cf. J. Didelon et al.,Biorheology 37, 2000: 409-416) using laser radiation with a wavelengthof 808 nm. A photoelectric cell measures the variation in the lighttransmitted through the suspension. For example, measurements are takenfor 10 minutes. The instrument provides one or more of the parametersa-e mentioned above.

According to a first modality, the osmotic fragility is measured on asample whose initial temperature is between +1 and +8° C., preferablywith distilled water also at this temperature, in conditions in whichtemperature variation does not affect the measurement. According to asecond modality, the osmotic fragility is measured on a samplemaintained at a temperature between +1 and +8° C. Thus, the measuringinstrument described in J. Didelon et al. (supra) can be modified topermit temperature regulation. Said temperature is preferably close toor equal to the lysis temperature.

Once one or more of these parameters have been determined, a relationcan be applied that takes into account said parameter or parameters inorder to determine either the flow rate of the cells in the dialyzer, orthe osmolarity of the dialyzate sufficient to obtain red blood cellsencapsulating the enzyme and/or the desired amount of the latter:Flow rate of erythrocytes=[A×(H ₅₀)]+[B×(V)]+K

-   -   A and B=variables that can be adapted in relation to the        dialyzer and the osmolarity of the lysis solution    -   K=constant for adjustment.        Osmolarity of dialyzate=[C×(H ₅₀)]+[D×(V)]+K    -   C and D=variables that can be adapted in relation to the        dialyzer and the flow rate of erythrocytes in the dialyzer    -   K=constant for adjustment.

According to one aspect of the invention, the lysis procedure is startedwhen the temperature of the suspension of erythrocytes is between +1 and+8° C., and the osmotic fragility has been measured and the lysisparameters have been recorded.

According to a preferred embodiment, the concentration of NaCl in g/Lwhich brings about 50% haemolysis is measured (parameter d.) and theflow rate of the erythrocyte suspension in the dialysis cartridge isadjusted in accordance with the measured concentration values.

According to an aspect of the invention, the lysis procedure is startedwhen the temperature of the erythrocyte suspension is from +1 to +8° C.,and the osmotic fragility has been measured and the lysis parametersrecorded.

According to an advantageous characteristic, the initial suspension tobe treated is placed in the lysis-internalisation chamber mentionedabove. According to one embodiment of the invention, the method employsa refrigerated module equipped with temperature control, and in thismodule is placed a bag of the suspension of erythrocytes refrigeratedbetween +1 and +8° C., already connected, or which is then connected, toa disposable sterile removable assembly, comprising a dialysiscartridge, tubes for connecting the cartridge to the bag and to thelysis solution, and in addition the module has means for providingcirculation of the suspension of erythrocytes and of the lysis solution,the temperature within said module being stabilized at a temperaturebetween +1 and +8° C. The refrigerated module is dimensioned so that itcan accommodate the bag and the disposable removable assembly. Thearrangement of the bag, the dialysis cartridge, and the lysis solution,connected together by the various tubes, within said single refrigeratedmodule is an advantageous characteristic of the method according to theinvention.

The term “bag” refers to the flexible bags or pouches commonly used inthe field of blood transfusion and blood derivatives.

According to an important aspect of the invention, steps are taken tomaintain the erythrocytes in homogeneous suspension in the bag, so as tomaintain a stable hematocrit value of the suspension passing through thedialyzer. According to a characteristic of the invention, the bag isaccordingly provided with external circulation in a loop, which providescirculation of the suspension from and to the bag.

“Dialysis cartridge” means an element comprising two compartmentsseparated by a dialysis partition, through which ion exchange can takeplace, enabling the osmotic pressure of an aqueous solution located inone of the compartments to be altered in a controlled manner byintroducing an aqueous solution containing a salt in the othercompartment. This type of cartridge is widely used in the medical field.According to a preferred modality, a hollow-fiber dialysis cartridge isused, for example having the following specifications: inside diameterof the fibers between 100 and 400 μm, total external surface of thefibers between 0.3 and 2 m², length of fibers between 10 and cm,coefficient of ultrafiltration between 1.5 and 8 ml/h.mmHg.

As already mentioned, the lysis procedure can be started when thetemperature of the suspension in the bag is between +1 and +8° C.According to an interesting modality, the temperature of the suspensionis controlled by means of a sensor located on the external loopcirculation.

Depending on the osmotic fragility determined, action can be taken ontwo main parameters, the flow rate of the suspension of erythrocytes inthe dialysis cartridge and the osmolarity of the lysis solution, itbeing preferable to set, in both cases, a constant flow rate for thelysis solution. The value of the flow rate is not critical. Typically,for a hollow-fiber dialysis cartridge as described above, the flow rateof the lysis solution is set between 50 and 300 ml/min, and preferablybetween 150 and 250 ml/min.

The lysis solution is a saline solution that is hypotonic relative tothe suspension of red blood cells. When it is set to a constant value,its osmolarity can typically be between 20 and 120 mOsm, preferablybetween 70 and 110 mOsm, for example of the order of 90 mOsm.

As an example, the lysis solution can comprise Na₂HPO₄ and/or NaH₂PO₄and a sugar such as glucose.

According to a first modality, the flow rate of the suspension oferythrocytes through the dialysis cartridge is adjusted, whereas theflow rate and osmolarity of the lysis buffer are fixed. Higher osmoticfragility means higher flow rate of the suspension. Typically, for acartridge with the specifications stated above, the flow rate will needto vary in the range from 5 to 200 ml/min, preferably from 10 to 40ml/min.

According to a second modality, the osmolarity of the lysis solution isadjusted, whereas the flow rates of the suspension and of the lysissolution are fixed. Higher osmotic fragility means increasing theosmolarity of the lysis solution. Typically, the osmolarity will need tovary in the range from 10 to 200 mOsm/l, preferably from 20 to 150mOsm/l.

According to a third modality, both the flow rate of the suspension oferythrocytes through the dialysis cartridge, and the osmolarity of thelysis solution are adjusted.

The enzyme to be encapsulated can be present in the bag of suspensionand/or can be introduced, preferably gradually, in the circulation ofsuspension upstream or downstream of the dialysis cartridge. As thevolumes introduced are small, refrigeration of the enzyme is optional.

Preferably, the suspension of red blood cells is produced from PRBCs ofa blood group compatible with the recipient, deleukocytized, withoutlisted pathogen, notably presented in a bag, for example of 500 ml. Thered blood cells may have been irradiated when they are intended forhighly immunodepressed patients liable to display a graft/host immunereaction (R. J. Davey, Immunol. Invest. 1995, 24 (1-2): 143-149).

According to a particular feature of the invention, the initial PRBCs,used for preparing the suspension, were treated beforehand to removeelements from the blood other than the erythrocytes. This type oftreatment, for example washing with a saline solution to remove theplasma or a preserving solution, is known by a person skilled in theart.

According to a particular modality, washing is carried out in thepresence of the enzyme to be encapsulated.

Washing can be carried out by any usual technique, such as thequadruple-bag or 4-bag technique for the washing of red blood cells(MacoPharma method and transfer bag). It is also possible to use anautomatic red blood cell washer of the type COBE 2991 Cell Processor.

According to another characteristic of the invention, the erythrocytescan be treated beforehand with a solution for increasing and/orhomogenizing their osmotic resistance. Such solutions are known by aperson skilled in the art. For example, a solution containingL-carnitine can provide an improvement of the osmotic resistance of thered blood cells. As other examples, we may mention solutions of heparin,of citrate-phosphate-dextrose (CPD) and of mannitol.

The temperature during the lysis stage is preferably maintained between+2 and +6° C., and even more preferably around +4° C.

The resealing process is preferably effected by heating the lysedsuspension and adding a hypertonic resealing solution. The resealingtemperature can be between +30 and +40° C. It is preferably between +35and +38° C., for example about 37° C. Incubation can typically last for15 to 45 minutes.

Preferably, the suspension leaving the dialysis cartridge as well as ahypertonic resealing solution are introduced, preferably continuously,into an intermediate bag. There the suspension is heated, and incubatedat the desired temperature for a sufficient time to ensure resealing.According to a particular aspect, the intermediate bag is placed in aheated module or container, the interior temperature of which isregulated to the chosen temperature.

As a variant, the suspension and the resealing solution are introducedinto an intermediate bag. When all of the suspension has been collectedin this bag it is sealed and transferred to a module for heating andincubation at the desired temperature.

The suspension of resealed red blood cells can then undergo one or morestages of washing with a saline solution, in order to remove cells thatwere poorly resealed or not resealed, residues and extracellularhemoglobin.

According to another characteristic, the erythrocytes are packaged in anerythrocyte storage solution, for example containing L-carnitine.

The erythrocytes produced are preferably stored at a temperature between+1 and +8° C., preferably between +2 and +6° C., typically at about +4°C.

The final hematocrit value of the product is preferably between 40 and80%, typically between 40 and 70%.

The present invention can be implemented using a lysis-resealing devicecomprising:

-   -   a module that can be refrigerated at a temperature between +1        and +8° C., comprising refrigerating means and temperature        regulating means,    -   a disposable sterile removable assembly, designed for fitting in        the module and comprising a dialysis cartridge that can be        connected on the one hand to a feed of lysis solution and on the        other hand to a feed of suspension of erythrocytes,    -   means for controlling the flow rate of the suspension of        erythrocytes through the lysis cartridge and/or adjusting the        osmolarity of the lysis solution, as a function of the osmotic        fragility of the erythrocytes to be treated.

According to one embodiment, the removable assembly is a disposable kitand comprises a bag for containing the suspension of erythrocytes and atube connecting said bag to the dialysis cartridge, and the modulecomprises a pump that works in conjunction with said tube to circulatethe suspension of erythrocytes from the bag to and through thecartridge, said pump being optionally coupled to flow regulating means.The assembly ensures that sterility is maintained.

According to an advantageous characteristic, the bag is additionallyequipped with a tube with both of its ends connected in a loop to thebag, and the module contains a pump that works in conjunction with saidtube to provide circulation of the contents of the bag from and to saidbag.

According to another advantageous characteristic, a temperature sensoris arranged on said loop of tube.

According to another characteristic, an enzyme injection tube isconnected to the tube connecting the bag to the “blood” inlet of thedialysis cartridge.

According to another characteristic, the dialysis cartridge is connectedby a tube to a bottle that can contain the lysis solution and therefrigerated module contains means for receiving said bottle and a pumpthat can operate in conjunction with said tube to circulate the lysissolution to and through the dialysis cartridge.

According to one characteristic, the refrigerating and temperaturecontrolling means are able to maintain a temperature between +2 and +6°C., and preferably of the order of +4° C. in the module.

According to another characteristic, the “blood” outlet from thedialysis cartridge is connected to an outlet tube leading, or which canlead, to the exterior of the module. According to anothercharacteristic, a tube for injection of active principle is connected tosaid outlet tube. The outlet tube can be connected to a second bag(intermediate bag) that is able to collect the suspension oferythrocytes after lysis as well as a resealing solution (preferablyintroduced via a secondary tube opening into the outlet tube a littleupstream of its opening into the intermediate bag). Said bag isadvantageously arranged in a second module equipped with means ofcontrolling the temperature in said module between +30 and +40° C.,preferably between +35° C. and +38° C.

According to an advantageous embodiment, the disposable removableassembly contains all of the following: the bags, circulation tubes,injection tubes (equipped with an injection device or a receptacle thatis intended to operate in conjunction with such a device), dialysiscartridge, and preferably a bottle of lysis solution.

Preferably, the removable assembly does not itself have specific meansintended for refrigeration or heating. These functions are only providedby the modules or chambers in which the two parts of the assembly areplaced.

The pumps used in the method and the device of the invention arepreferably peristaltic pumps; according to one embodiment, the pumpproviding recirculation of the suspension from and to the initial bagand the pump for circulating the lysis buffer have a predeterminedconstant rotary speed, whereas the pump sending the suspension to thedialysis cartridge has a rotary speed that is controllable as a functionof the osmotic fragility of the erythrocytes to be treated.

The enzyme can be introduced by any suitable means, for example afixed-flow syringe pump, optionally driven, connected to thecorresponding injection tube. As a variant, the syringe pumps can bereplaced with peristaltic pumps.

The device includes means of controlling the flow rate of the suspensionof erythrocytes through the lysis cartridge and/or adjustment of theosmolarity of the lysis solution, as a function of the osmotic fragilityof the erythrocytes to be treated.

According to one characteristic, the flow regulating means are designedto control the pump sending the suspension to the dialysis cartridge.According to another, alternative characteristic, the regulating meansare designed for regulating the osmolarity of a lysis solution, eitherby dilution to lower the osmolarity, or to increase said osmolarity byintroducing a suitable solute. As a variant, a lysis solution ofosmolarity adjusted to the osmotic fragility of the erythrocytes to betreated is introduced into the module.

According to a preferred modality, the device comprises electronic meansfor controlling the lysis process and optionally the resealing process,in accordance with instructions entered by the operator (e.g. dataconcerning the flow rate of the suspension of erythrocytes are enteredby the operator directly), or in accordance with data entered by theoperator, relating to the osmotic fragility (the electronic means thenbeing designed for determining and adjusting the lysis parameters, e.g.the flow rate of the suspension of erythrocytes). Said electronic meansare preferably connected to temperature sensors (for controlling thetemperature in the modules and/or at the temperature sensor for thesuspension of erythrocytes). Said means can control and operate thepumps, for example the pressure and the flow rate of the suspensionthrough the dialysis cartridge.

Preferably, the modules are equipped, on one face at least, with a glasssurface, for visual control of the installation and the circulation ofthe solutions and suspensions.

The invention will now be described in more detail on the basis ofembodiments that are used as non-limiting examples, referring to thedrawings in which:

FIG. 1 is a schematic representation of a lysis-resealing deviceaccording to the invention;

FIG. 2 is a flow chart of the method;

FIG. 3 is a graph illustrating the arginine versus citrullineconcentrations in the supernatant of red blood cells with or without ADI(arginine deiminase);

FIGS. 4 and 5 represent graphs relative to the pharmacokinetics in thered blood cells for arginine and citrulline concentrations; FIG. 4 showsthe variation in arginine concentration during time (up to 48 hoursafter treatment) and FIG. 5 shows the variation in citrullineconcentration during time (up to 48 hours after treatment) for threegroups of mice treated with: ADILE (arginine deiminase loaded red bloodcells), ADI (free arginine deiminase)+RBC, or RBC.

RBC or BC is used herein to designate red blood cells.

EXAMPLE 1 Installation

Reference will first be made to FIG. 1. A first box shown by dashedlines depicts a first module 1, having an overall shape of aparallelepiped, with a glass-covered front (not shown), arranged so thatit can be opened and closed. At the back of this module there areperistaltic pumps P1, P2 and P3, and means, not shown, for receiving aremovable assembly that will now be described. Pumps P1 and P3 have apredetermined, constant delivery. Pump P2 is controlled so that itsdelivery varies.

The removable assembly includes a bag 2 that is flexible in volume,containing a suspension of erythrocytes to be lysed. Said bag 2 isequipped with a flexible tube 3, in a loop, operating in conjunctionwith pump P1, to provide circulation from and to the bag to maintain theerythrocytes in suspension. Said bag is in addition connected at itsbase to a flexible tube connected to the inlet of the “blood”compartment of a dialysis cartridge 5. Said tube 4 operates inconjunction with pump P2, which provides circulation of the suspensionfrom the bag to the cartridge. A driven syringe pump PS1 is connected totube 4 upstream of cartridge 5, and said syringe pump permits the enzymeto be introduced into the circulation of erythrocytes. The outlet of the“blood” compartment of cartridge is connected to an outlet flexible tube6, which opens onto the exterior of module 1. A second driven syringepump PS2 is connected to tube 6, and this syringe pump permits theenzyme to be introduced into the circulation of lysed erythrocytes. Abottle 7 containing a lysis solution is arranged in module 1, and isconnected to the “dialyzate” inlet of cartridge 5 by a flexible tube 8,which operates in conjunction with pump P3 to provide circulation of thelysis solution through cartridge 5. Finally, the lysis solution leavingthe cartridge is removed from module 1 by a flexible discharge tube 9,which ends in a bottle 10 located outside of module 1.

Outlet tube 6 goes into a second module 11 with the overall shape of aparallelepiped, with a glass-covered front (not shown), arranged so thatit can be opened and closed. At the back of this module there are means,not shown, for receiving elements forming part of the removable assemblythat has just been described partially. Said elements comprise aflexible bag 12, connected to tube 6, and in which the lysed suspensionwill be stored. A driven syringe pump PS3 is connected to tube 6, forinjecting the resealing product.

The removable assembly is made entirely of flexible, transparentplastic, so that the process is completely visible.

The device is further provided with various means that are not shown:

-   -   means for cooling the interior of module 1 and regulating its        temperature between +2 and +4° C., comprising, among other        things, a temperature sensor located on tube 3 for measuring the        temperature of the suspension circulating therein, and a        temperature sensor for measuring the temperature T1 inside        module 1,    -   module 11 is further provided with means of heating the interior        of module 11 and regulating the temperature T2 therein between        +37 and +38° C.; a temperature sensor is fitted inside the        module.    -   means (for example ultrasonic or colorimetric) for detecting the        presence of erythrocytes in the tubes, at D1 and D2,    -   means PR1 for measuring the pressure at the inlet of the        dialysis cartridge.    -   electronic device receiving on the one hand data arriving from        the temperature and pressure sensors and the detecting means,        and on the other hand data relating to the settings of the lysis        parameters; on the basis of said data, the device controls pumps        P1, P2 and P3. A process flow chart is shown in FIG. 2.

The electronic device comprises a computer, designed for executing theabove flow chart.

Implementation of said device leads to the recovery, at 12, of a bagcontaining a suspension of erythrocytes containing the enzyme.

EXAMPLE 2 Production of Erythrocytes Encapsulating Arginine Deiminase

400 ml of blood is taken from the patient. The blood, maintained at 4°C., is deleukocytized and washed with a saline solution to remove theplasma, and placed in a flexible bag with a volume of 250 ml, at ahematocrit adjusted to 80%.

An aqueous solution of arginine deiminase is added to the suspension oferythrocytes so as to obtain a concentration of 400 IU of enzyme per mland a hematocrit of 70%.

Take 1 ml of the suspension at 4° C. and place it in the instrument formeasuring osmotic fragility described in J. Didelon et al. 2000 citedpreviously, the operating principle of which was described above.Measurements are taken for 10 minutes. The instrument makes it possibleto determine the salinity that gives 50% hemolysis. This salinity isgenerally between 3 and 5.5 g NaCl per liter.

The 250-ml capacity flexible bag 2, containing the suspension oferythrocytes and the enzyme, is placed in the installation of Example 1,and the suspension and the hypotonic lysis solution are admittedgradually into the respective compartments of the dialysis cartridge.The flow rate of the suspension of erythrocytes in the dialyzer iscontrolled between 15 and 30 ml/min, as a function of the salinityparameter determined in the preceding stage (osmotic fragility orresistance).

The resealing solution is added in line at 10% v/v to the suspension oflysed erythrocytes just upstream of bag 12. The suspension is incubatedfor 30 min at 37° C. in the bag. It is then washed with a salinesolution, a preserving solution is added to it (SAG-mannitol), then thebag is stored at +4° C. until it is used.

The method makes it possible to obtain erythrocytes having an enzymaticactivity between 80 and 180 IU per ml of pure erythrocytes.

The total volume of the suspension is administered to the patient byintravenous perfusion in accordance with the usual practice of bloodtransfusion.

EXAMPLE 3 In Vitro Assay

Arginise deimainase is an arginase catabolizing enzyme transformingarginine into citrulline and ammoniac. The aim of the study was toobserve and confirm the depletion activity of arginine deiminaseobtained from Pseudomonas aeruginosa once encapsulated into red bloodcells. In this purpose, arginine deiminase-loaded red blood cells wereincubated with arginine containing buffer. Citrulline and argininelevels were subsequently assessed by HPLC MS MS method.

Preparation of Arginine Deiminase Loaded Red Blood Cells (RBC)

Solution of recombinant SeMet-containing L-Arginine deiminase (ADI) (120Ul/ml) originated from Pseudomona aeruginosa.

Fresh heparinized OF1 mouse blood was obtained from Charles Riverlaboratories and centrifuged (800 g. 10 min at 4° C.) to remove plasmaand buffy coat. Packed erythrocytes were washed 3 times (1:1 v/v) withNaCl 0.9% (800 g, 10 min at 4° C.). After the final washing erythrocyteswere mixed with (CGR-ADI) or without (CGR-LR) 20 Ul/ml argininedeiminase and the haematocrit of the RBC suspension was adjusted to 70%(using ADI solution or saline).

Lysis of erythrocytes was obtained by a continuous flow dialysis processinto a dialysis bag (cutoff 10 Kd). The hypotonic step was performed at4° C. during 60 minutes against an adequate volume of lysis buffer at 40mOsm/1 (NaH₂PO₄, 2H₂O 0.73 g/l; Na₂HPO₄, 12H₂O 5.035 g/l; glucose 0.36g/l). 100 ml of hypotonic solution were added for 1 ml of erythrocytesintroduced. After collection, the suspension of lysed erythrocytes wasincubated at 37° C. for 10 min. The cells were then resealed andannealed by incubation at 37° C. during 30 minutes in a 1/10 (v/v) ofresealing solution (adenine 0.39 g/l; inosine 15.6 g/l; sodium pyruvate6.4 g/l; NaH₂PO₄, H2O 4.9 g/l; NaHPO₄, 12H₂O 10.9 g/l, glucose 11.5 g/l;NaCl 50 g/l). After resealing the erythrocytes were washed 3 times (800g, 10 min at 4° C.) in Tris 310 mOsm/1 pH 7.4, BSA 4%.

Whole blood, RBC suspension before and after dialysis step weremonitored for haematocrit (Ht), mean cell volume (MCV), mean cellhaemoglobin (MCHC) and mean corpuscular haemoglobin concentration (MCHC)using a Cobas Micros 601 CS 14/12 cell counter. CGR-ADI and CGR-LRsuspension were at 25% haematocrit after dialysis.

Aliquots of RBC suspension (with or without ADI) were collected beforeand after dialysis step for subsequent ADI activity measurement.

Assay of ADI Activity

Assay of ADI activity was performed on aliquots of RBC suspension(CGR-ADI and CGR-LR) collected before and after dialysis.

Haematocrit of CGR-ADI and CGR-LR aliquots before dialysis were adjustedfrom 70% to 40% by an adequate dilution in NaCl 0.9%. Haematocrit ofCGR-ADI and CGR-LR aliquot suspension after dialysis was of 25% andwasn't modified. The rate of ADI encapsulated was determined bymeasurement of ADI activity in whole blood or in supernatant. Todetermine ADI activity in whole blood, one third of RBC suspensionaliquot was frozen in liquid nitrogen during 5 minutes and warmed at 37°C. and 10 μl of a 10 fold dilution in 50 mM MES of frozen RBC was thenused for enzymatic assay activity. To determine the enzymatic activityoutside red cells, the other two-third of RBC suspension werecentrifuged at 4° C. during 10 minutes and 10 μl of a 2 fold dilution in50 mM MES of supernatant was used for enzymatic assay. The amount ofcitrulline formed in 10 min was quantified by the colorimetric assay ofPrescott and Jones. The standard assay mixture contained 900 μl MESbuffer 0.1M pH 6.0, MgCl₂ 20 mM and 10 μl of supernatant or frozen RBCsample. The reaction was started by addition of 1 ml 10 mM L-arginineand was allowed to continue for 5, 10, 15 or 20 min at 37° C. Thereaction was stopped at theses different times by the addition of 1 mlof an antipyrine-diacetylmonoxime solution. The mixture was boiledduring 20 minutes and the absorbance at 466 nm was measured. Standardcurves were constructed by appropriately diluting a stock solution ofcitrulline. Activity of ADI between 15 and 20 min was defined asmicromoles of citrulline formed per min of enzyme. All measures weredone in duplicates.

In Vitro Functionality Assay

In vitro assay was completed by incubating arginine deiminase-loaded redblood cells (CGR-ADI) in a buffer containing arginine and observing thelevels of both arginine and citrulline aminoacid. Control of thereaction was realized with red blood cells loaded without argininedeiminase (CGR-LR) incubated in the same conditions.

1 ml of prewarmed arginine deiminase-loaded red cells (CGR-ADI) weremixed with 1 ml of buffer containing 300 μM arginine, 20 mM MgCl₂ inTris pH 7.4. Tris buffer was prepared at 320 mOsm/kg, pH 7.4. Thecontrol assay was performed by mixing in the same conditions anequivalent amount of CGR-LR with arginine containing buffer. Mixing wasrealized by upside down movements. 400 μl of sample, representating time0, were immediately collected in an ependorf tube and placed at 4° C.The rest of the mixture was incubated during 30 minutes at 37° C. At theend of the reaction, 400 μl representing time 30 (for 30 minutes) werecollected in an ependorf and placed at 4° C. For all the samples of 400μl collected, three quarter (300 μl) were centrifuged at 4° C. during 10min. After centrifugation, an aliquot of 100 μl of supernatant wascollected and frozen at −20° C. The other quarter was directly frozen inliquid nitrogen during five minutes. After warming at 37° C., an aliquotof 50 μl of sample was collected and frozen at −20° C.

Arginine and citrulline levels in each sample were then assessed byHPLC/MS/MS method.

Results: see FIG. 3

Within 30 minutes at 37° C., the RBC entrapping ADI are able to depletethe arginine contained in the medium (supernatant) from about 135 μmol/Lto about 42 μmol/L. In the same time, citrulline concentration in themedium (extra-erythrocytes) is produced from about 17 to about 110μmol/L. The activity of ADI in the supernatant was under the limite ofdetection (<0.1 Ul/Ml). The activity measured in the RBC pellet was 2.56Ul/mL. It proves that the arginine deimination is provided by the intraerythrocyte ADI, and that arginine enter into the erythrocyte to bedigested into them. In addition, it strongly suggests the citrullineproduced into the erythrocyte is going to the extra RBC medium thoughthe red cell membrane.

Concerning the Control RBCs, which are processed erythrocytes where ADIis replaced by saline, arginine from the extra RBC medium decreases onlyfrom about 165 μmol/L to about 157 μmol/L within 30 minutes at 37° C.,and citrulline reachs only from about 1 to about 2 μmol/L. This lowdepletion can be explained by the endogenous activity of arginasecontained into the RBCs. We underlined again arginase do not producecitrulline while arginine digestion, which is specific to ADI.

EXAMPLE 4 Kinetic Study of Arginine Concentration in Mice Plasma inResponse to Injections of Two Formulations of Arginine Deiminase

The aim of the study was to follow the plasma pharmacokinetic ofarginine and citruline in OF1 mice in response to injection of argininedeiminase-loaded erythrocytes

Preparation of Test and Control Substances

Recombinant SeMet-containing L-Arginine deiminase (ADI) (120 UI/ml)originated from Pseudomona aeruginosa.

Free arginine deiminase (ADI+RBC) was diluted in washed mouse red bloodcells (RBC) and Sag-mannitol 1/3 (v/v) (Haemonetics) in order to obtaina final concentration at 10 UI/ml of packed RBC at hematocrit of 50%.Sag-mannitol was supplemented with 10 mM MgCl₂ prior to addition.

Test substance (ADILE) consisted in arginine deiminase loaded red bloodcells (RBC). The procedure of preparation of arginine deiminase loadedred blood cells was determined as described for in vitro functionalitytest of ADI. Before dialysis ADI was mixed with washed packederythrocytes in order to have a final concentration 50 UI/ml. Afterdialysis, encapsulated RBC were mixed with Sag-Mannitol 1/3 (v/v)supplemented with 10 mM MgCl₂. Final hematocrit was adjusted to 50%. TheADI activity obtained after encapsulation was of 8.35 UI/ml ofencapsulated RBC at hematocrit of 50%.

A third sample with no enzyme (CGR) was prepared with washed RBCresuspended with Sag Mannitol (supplemented with 10 mM MgCl₂) at finalhematocrit of 50%. Saline solution was added in replacement of ADI.

During whole experiment, whole blood, RBC suspension before and afterdialysis step were monitored for haematocrit (Ht), mean cell volume(MCV), mean cell haemoglobin (MCHC) and mean corpuscular haemoglobinconcentration (MCHC) using a Cobas Micros 601 CS 14/12 cell counter.Aliquots of RBC suspension were collected before and after dialysis stepfor subsequent ADI activity measurement. ADI activity measurement wasdetermined as described previously.

Animals

64 OF1 female mice, 5-6 week-old and weighing 18-22 g were obtained fromCharles River Laboratories (L'Arbresle, France). Animals were observedfor 7 days in a specific-pathogen-free (SPF) animal care beforetreatment. The experimental protocols were approved by the FrenchMinistries of Agriculture and Research. The 64 healthy OF1 mice wererandomized in 1 group of 4 mice and 3 groups of 20 mice. Treated micereceived a single injection by intravenous route in an injection volumeof 250 μl.

Treatment Schedule

The treatment schedule was chosen as followed: mice from group 1 werenot treated; mice from group 2 were treated by mouse red blood cellswashed at hematocrit of 50% (RBC); mice from group 3 received a singleinjection of arginine deiminase loaded-erythrocytes (ADILE); mice ofgroup 4 received a single injection of free arginine deiminase (ADI+RBC)in suspension in mouse RBC washed at hematocrit of 50%. The differentproduct samples were administered in double blind.

After treatment, mice were sacrificed by cardiac puncture. IsofluraneForene (Centravet, Bondoufle, France) was used to anaesthetize the micebefore sacrifice. The sacrifice of mice was performed as described belowin the table. Approximately 800 μl of whole blood were collected inheparin lithium glass tubes and kept immediately in ice-water bath aftercollection. Blood samples were immediately centrifuged at 2,500 g for 10min at +4° C. to obtain plasma. About 200 μl of plasma were transferredinto propylene tubes, immediately frozen at −20° C. The remaining bloodcell pellet was transferred into propylene tubes, immediately frozen at−20° C. until analysis. The levels of arginine and citrulline weremeasured in one vial of plasma and blood cell pellet.

Amino Acids Analyses

Concentration of arginine and citrulline in plasma and blood cellpellets were measured after extraction of arginine and citrulline byHPLC/MS/MS method.

Dose ADI Administration Sampling times Treatments (UI/kg) route (hours)No mice None 0 NA NA 4 RBC 0 IV 3 4 (10 ml/kg) 6 4 12 4 24 4 48 4 ADILE100 IV 3 4 (10 ml/kg) 6 4 12 4 24 3 48 3 ADI + RBC 100 IV 3 4 (10 ml/kg)6 4 12 4 24 4 48 4 NA: Not ApplicableResults: see FIGS. 4-5

-   -   1) considering the dosage of arginine in a red blood cell        pellet, it is observed a strong and rapid decrease in        concentration in the mice which received ADI free or entrapped.        No significant modification was observed in mice who received        normal RBC. However, within 12 hours the concentration in        arginine come back to the normal values for the ADI free group        (ADI+RBC) and is maintained very low during at least 48 hours        for the Entrapped ADI (ADILE).    -   2) In the same time arginine is depleted, citrulline is        produced. However, it is observed while the high level is        maintained for ADILE up to 48 hours, it come back to the normal        value within 24 hours for the ADI+RBC group. No significant        modification was observed in mice who received normal RBC.

This prove the entrapment of ADI into RBC is possible by lysis/resealingsteps, and that the ADI loaded into RBC is much longer efficient thanADI in free solution.

What is claimed is:
 1. A pharmaceutical composition comprisingerythrocytes encapsulating arginine deiminase (ADI) in suspension in apharmaceutically acceptable saline solution, wherein the encapsulationconfers prolonged in vivo half-life beyond 24 hours to the argininedeiminase encapsulated in the erythrocytes, wherein the hematocrit inthe suspension is from 40% to 70%, wherein the volume of the suspensionis from 10 to 250 ml, and wherein the erythrocytes contained in thecomposition have an ADI enzymatic activity of from 16.7 to 180 IU permilliliters of pure erythrocytes.
 2. The pharmaceutical composition ofclaim 1, wherein the saline solution comprises NaCl and at least oneingredient selected from the group consisting of glucose, dextrose,adenine, mannitol, and a mixture thereof.
 3. The pharmaceuticalcomposition of claim 1, wherein the saline solution is SAG-mannitol. 4.The pharmaceutical composition of claim 1, wherein the saline solutionis ADsol.
 5. The pharmaceutical composition of claim 1, furthercomprising a preservative for erythrocytes.
 6. The pharmaceuticalcomposition of claim 5, wherein the preservative is L-carnitine.
 7. Thepharmaceutical composition of claim 1, wherein the hematocrit of thesuspension of erythrocytes is between 45 and 55%.